Anthrax toxin is a three-part toxin secreted by Bacillus anthracis consisting of protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa) (Smith et al., J. Gen. Microbiol., 29:517-521 (1962); Leppla, Sourcebook of bacterial protein toxins, p. 277-302 (1991); Leppla, Handb. Nat. Toxins, 8:543-572 (1995)), which are individually non-toxic. The mechanism by which individual toxin components interact to cause toxicity is reviewed in Leppla, Handb. Nat. Toxins, 8:543-572 (1995). Protective antigen, the central component of the Anthrax toxin system, is cleaved at the sequence RKKR167 (SEQ ID NO:15) by cell surface furin or furin-like proteases (Klimpel et al., Proc. Natl. Acad. Sci. USA, 89:10277-10281 (1992); Molloy et al., J. B. Chem., 267:16396-16402 (1992)) into two fragments: PA63, a 63 kDa C-terminal fragment, which is capable of forming oligomers of multiple PA63 units; and PA20, a 20 kDa N-terminal fragment, which is released and plays no further part in the Anthrax toxin scheme (Klimpel et al., Mol. Microbiol., 13:1094-1100 (1994)). Dissociation of PA20 allows PA63 to form heptamers (Milne et al., J. Biol. Chem., 269:20607-20612 (1994); Benson et al., Biochemistry, 37:3941-3948 (1998)) and octamers (Kintzer et al., J. Mol. Biol., 392:614-629 (2009)) that bind LF or EF (Leppla et al., Bacterial protein toxins, p. 111-112 (1988)), and the resulting hetero-oligomeric complex is internalized by endocytosis (Gordon et al., Infect. Immun., 56:1066-1069 (1988)).
Recognizing the importance of protective antigen activation by proteolytic cleavage, researchers have in the past decade or so engineered several modified versions of protective antigen mutants to replace the native furin cleavage site with the recognition site of anther pre-selected proteinase in order to specifically target cells that overexpress the proteinase for delivery of an effector molecule, such as lethal factor or edema factor. For example, mutant protective antigen proteins have been made to contain cleavage site recognized by matrix metalloproteinases (MMPs) and proteases of the plasminogen activation system (such as tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA), see, e.g., Romer et al., APMIS 107:120-127 (1999)), which are typically overexpressed in tumors, such that the modified Anthrax toxin systems can be used in a tumor-specific delivery scheme. See, e.g., WO01/21656 and WO2008/076939.
Since these modified Anthrax toxin systems rely on the overexpression of just one proteinase to target a cell population, undesired side effects stemmed from non-specific cytotoxicity have been a concern. Although efforts have been made to enhance target specificity and reduce non-specific cytotoxicity (see, e.g., Liu et al., Nature Biotech. 23(5):725-730 (2005)), there remains a pressing need to further improve the Anthrax toxin delivery system for even higher delivery efficiency and lower side-effect toxicity. The present invention fulfills this and other related needs.